Corneal HSV-1: LAT's Anti-Apoptosis Activity and Latency

复发性HSV-1感染作为病毒再激活的结果是病毒诱导的失明的主要原因。 我们的总体目标是阐明HSV-1潜伏再激活背后的分子机制 周期，希望导致开发一种减少HSV-1再活化的方法，从而减少HSV-1感染的发生率。 HSV-1诱导角膜盲。LAT是唯一在潜伏期大量转录的病毒基因， 通过阻断细胞凋亡再激活表型。由于没有LAT蛋白的报道，LAT被认为 通过非编码RNA发挥作用。与此相反，我们现在有强有力的证据表明， LAT蛋白（L2）在潜伏期期间表达。我们也有证据表明，第一个小LAT RNA（RNA-1） 似乎也有抗凋亡活性。由于LAT的抗凋亡活性是其最重要的作用， 由于L2和RNA-1都表现出抗凋亡活性， 通过LAT的功能性第一个1.5 Kb，我们假设两者都有助于LAT增强 再活化表型我们追求这些创新发现的具体目标包括： 1.确认L2（由nts 487-669编码）和剪接L2是真实LAT蛋白的假设 a）人源化L2 ORF nt序列而不改变L2氨基酸 表达LAT功能性第1个1.5 Kb的质粒中的序列，并确认质粒的保留 抗凋亡活性; B）表达人源化L2（h-L2）ORF（60 aa）相对于h-剪接的-L2 ORF（118 aa） 在没有LAT侧翼序列的质粒中，并确认它们的抗凋亡活性; L2末端和与myc剪接的-L2，并测定它们在不同野生型病毒中的变化， 急性感染期间的表达与潜伏感染与再激活。 2.证实L2、剪接的L2和RNA-1有助于LAT支持WT的能力的假设。 再激活表型，并且其中至少一个在再激活期间发挥其主要影响，而不是 潜伏期的建立：a）构建敲除突变体并确认其降低的再活化 表型; B）构建仅表达h-L2、h-剪接的-L2或RNA-1的突变体，并证实它们 与LAT（-）病毒相比具有增加的再活化表型; c）阻断L2的表达/功能， 剪接的L2，或RNA-1在建立潜伏期与再激活，并确定对 再活化表型 3.验证L2、剪接L2和RNA-1通过不同机制干扰细胞凋亡的假设 通过：a）证实它们具有不同的阻断由不同试剂/方法诱导的细胞凋亡的能力; B）证实它们阻断外源性Fas途径中的不同步骤; 凋亡因子，表明它们阻断结合的凋亡因子的功能。

Recurrent HSV-1 infection as a result of viral reactivation is a major cause of viral induced blindness. Our overall goal is elucidation of the underlying molecular mechanisms behind the HSV-1 latency-reactivation cycle, hopefully leading to development of a means for reducing HSV-1 reactivation and hence the incidence of HSV-1 induced corneal blindness. LAT, the only viral gene abundantly transcribed during latency enhances the reactivation phenotype by blocking apoptosis. Since no LAT protein has been reported, LAT is thought to function via a noncoding RNA. In contrast to this notion, we now have strong evidence for an anti-apoptotic LAT protein (L2) that is expressed during latency. We also have evidence for the first small LAT RNA (RNA-1) that also appears to have anti-apoptosis activity. Since LAT's anti-apoptosis activity is its most important latency related function and since L2 and RNA-1 both appear to have anti-apoptosis activity and are encoded by the functional 1st 1.5 Kb of LAT, we hypothesize that both contribute to LAT's ability to enhance the reactivation phenotype. Our Specific Aims to pursue these novel innovative findings include: 1. Confirm the hypotheses that L2 (encoded by nts 487-669) and spliced-L2 are authentic LAT proteins with anti-apoptosis activity by: a) Humanizing the L2 ORF nt sequence without changing the L2 amino acid sequence in a plasmid expressing the functional 1st 1.5 Kb of LAT and confirming retention of the plasmid's anti-apoptosis activity; b) Expressing the humanized L2 (h-L2) ORF (60 aa) vs. the h-spliced-L2 ORF (118 aa) in plasmids with no LAT flanking sequences and confirming their anti-apoptosis activity; c) Tagging the C- terminus of L2 and spliced-L2 with myc in separate otherwise wt viruses and determining changes in their expression during acute infection vs. latent infection vs. reactivation. 2. Confirm the hypothesis that L2, spliced-L2, and RNA-1 contribute to LAT's ability to support the wt reactivation phenotype and that at least one of them exerts its main influence during reactivation rather than establishment of latency by: a) Constructing knock out mutants and confirming their reduced reactivation phenotype; b) Constructing mutants that express just h-L2, h-spliced-L2, or RNA-1 and confirming that they have an increased reactivation phenotype compared to LAT(-) viruses; c) Blocking expression/function of L2, spliced-L2, or RNA-1 during establishment of latency vs. reactivation, and determining the effect on the reactivation phenotype. 3. Test the hypothesis that L2, spliced-L2, and RNA-1 interfere with apoptosis via different mechanisms by: a) Confirming that they have differing abilities to block apoptosis induced by different agents/methods; b) Confirming that they block different steps in the extrinsic Fas pathway; c) Determining if they bind to different apoptotic factors, suggesting that they block function of the bound apoptotic factor.